Abstract The transcriptional activator TonEBP is essential for the development and function of the renal medulla. Activated by hypertonicity (hyperosmotic salinity), TonEBP is a key regulator for the urinary concentration via stimulating transcription of aquaporin-2 water channel and UT-A urea transporters. In addition, TonEBP is the master regulator for protecting the renal medullary cells from the deleterious effects of hypertonicity and urea via stimulating cellular accumulation of organic osmolytes and molecular chaperone. Genetically modified mice deficient in the renal TonEBP display severe medullary atrophy due to massive cell death, and life threatening volume depletion due to impaired ability to concentrate the urine. In patients, TonEBP is implicated in diseases including diabetic nephropathy, inflammation, and cancer metastasis. Despite the importance of TonEBP in health and disease, little is understood how changes in tonicity is recognized and the information is conveyed to TonEBP. Our data obtained in the previous period reveal clues that TonEBP itself is a tonicity sensor that controls the nuclear trafficking in response to changes in ambient tonicity. The data also demonstrate that TonEBP is covalently modified with acetyl groups and small ubiquitin-like modifiers (SUMO) in response to hypertonicity. In this proposal, we will pursue these exciting findings to understand the cellular signaling to TonEBP at the molecular level. We will explore two models of hypertonicity signaling. The intrinsic model states that TonEBP itself is a tonicity sensor (Aim 1). The extrinsic model states that a sensor signals to TonEBP via posttranslational modifications involving acetyl group and SUMO (Aim 2 and Aim 3). In Aim 1, we will uncover molecular basis of the tonicity sensing by investigating tonicity-responsive changes in the interaction between the nuclear localization signal of TonEBP and the cellular machinery carrying out the nuclear import. Aim 2 is to understand how the histone deaceytlase SIRT1 control the nuclear export of TonEBP in a tonicity-responsive manner. Sites of acetylation and regulation by hypotonicity will be delineated. Aim 3 is to understand how SUMO conjugation modulates the activity of TonEBP by examining transcriptional repression and protein stability. Cellular mechanism of the tonicity-dependent SUMO modification will be delineated. Project Narrative The proposed studies are likely to uncover fundamentally new information about how the TonEBP molecule is controlled -turned on or off. This information will provide new opportunity to develop therapy for renal diseases such as diabetic nephropathy and acute renal failure.